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Recent updates in the classification of myeloid neoplasms (MNs) recognize the poor prognostic impact of TP53 mutations, with particular emphasis on the TP53 allele status. Studies on the effect of TP53 allele status exclusively in therapy-related MNs (t-MNs) are lacking. We compared the clinicopathologic and survival characteristics of t-MNs with single-hit (SH) and multi-hit (MH) TP53 mutations. A total of 71 TP53-mutated t-MNs were included, including 56 (78.9%) MH and 15 (21.1%) SH. Both groups showed comparable genetic profiles with an excess of high-risk karyotypes and a paucity of other co-mutated genes. TP53 was the sole detectable mutation in 73.3% of SH and 75.0% of MH cases. The overall survival (OS) of SH TP53-mutated t-MNs was not significantly different from MH cases (median survival: 233 vs.273 days, p = 0.70). Our findings suggest that t-MNs with SH TP53 mutations share the poor prognostic and biologic profile of their MH counterparts. [ABSTRACT FROM AUTHOR]

Mitotically stable random monoallelic gene expression (RME) is documented for a small percentage of autosomal genes. We developed an in vivo genetic model to study the role of enhancers in RME using high-resolution single-cell analysis of natural killer (NK) cell receptor gene expression and enhancer deletions in the mouse germline. Enhancers of the RME NK receptor genes were accessible and enriched in H3K27ac on silent and active alleles alike in cells sorted according to allelic expression status, suggesting enhancer activation and gene expression status can be decoupled. In genes with multiple enhancers, enhancer deletion reduced gene expression frequency, in one instance converting the universally expressed gene encoding NKG2D into an RME gene, recapitulating all aspects of natural RME including mitotic stability of both the active and silent states. The results support the binary model of enhancer action, and suggest that RME is a consequence of general properties of gene regulation by enhancers rather than an RME-specific epigenetic program. Therefore, many and perhaps all genes may be subject to some degree of RME. Surprisingly, this was borne out by analysis of several genes that define different major hematopoietic lineages, that were previously thought to be universally expressed within those lineages: the genes encoding NKG2D, CD45, CD8α, and Thy-1. We propose that intrinsically probabilistic gene allele regulation is a general property of enhancer-controlled gene expression, with previously documented RME representing an extreme on a broad continuum.

Within the pancreas, Keratin 19 (KRT19) labels the ductal lineage and is a determinant of pancreatic ductal adenocarcinoma (PDAC). To investigate KRT19 expression dynamics, we developed a human pluripotent stem cell (PSC)‐based KRT19‐mCherry reporter system in different genetic backgrounds to monitor KRT19 expression from its endogenous gene locus. A differentiation protocol to generate mature pancreatic duct‐like organoids was applied. While KRT19/mCherry expression became evident at the early endoderm stage, mCherry signal was present in nearly all cells at the pancreatic endoderm (PE) and pancreatic progenitor (PP) stages. Interestingly, despite homogenous KRT19 expression, mCherry positivity dropped to 50% after ductal maturation, indicating a permanent switch from biallelic to monoallelic expression. DNA methylation profiling separated the distinct differentiation intermediates, with site‐specific DNA methylation patterns occurring at the KRT19 locus during ductal maturation. Accordingly, the monoallelic switch was partially reverted upon treatment with a DNA‐methyltransferase inhibitor. In human PDAC cohorts, high KRT19 levels correlate with low locus methylation and decreased survival. At the same time, activation of oncogenic KRASG12D signalling in our reporter system reversed monoallelic back to biallelic KRT19 expression in pancreatic duct‐like organoids. Allelic reactivation was also detected in single‐cell transcriptomes of human PDACs, which further revealed a positive correlation between KRT19 and KRAS expression. Accordingly, KRAS mutant PDACs had higher KRT19 mRNA but lower KRT19 gene locus DNA methylation than wildtype counterparts. KRT19 protein was additionally detected in plasma of PDAC patients, with higher concentrations correlating with shorter progression‐free survival in gemcitabine/nabPaclitaxel‐treated and opposing trends in FOLFIRINOX‐treated patients. Apart from being an important pancreatic ductal lineage marker, KRT19 appears tightly controlled via a switch from biallelic to monoallelic expression during ductal lineage entry and is aberrantly expressed after oncogenic KRASG12D expression, indicating a role in PDAC development and malignancy. Soluble KRT19 might serve as a relevant biomarker to stratify treatment. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

CEBPA variants are frequently recurring in acute myeloid leukemia (AML). The prognostic significance of CEBPA mutations has recently undergone a major shift in the 5th edition of WHO classification of hematological neoplasms and ELN 2022 classification. Whereas prior iterations did not specify the type of CEBPA mutation, the updated schema specify that only mutations localized to the C-terminal basic zipper (bZIP) domain are considered prognostically favorable. This change is based primarily on three recently published large datasets evaluating the prognostic significance of mutation location in CEBPA mutant AML. Here, we review the evolution of the prognostic classification of CEBPA variants.

Mitotically stable random monoallelic gene expression (RME) is documented for a small percentage of autosomal genes. We developed an in vivo genetic model to study the role of enhancers in RME using high-resolution single-cell analysis of natural killer (NK) cell receptor gene expression and enhancer deletions in the mouse germline. Enhancers of the RME NK receptor genes were accessible and enriched in H3K27ac on silent and active alleles alike in cells sorted according to allelic expression status, suggesting enhancer activation and gene expression status can be decoupled. In genes with multiple enhancers, enhancer deletion reduced gene expression frequency, in one instance converting the universally expressed gene encoding NKG2D into an RME gene, recapitulating all aspects of natural RME including mitotic stability of both the active and silent states. The results support the binary model of enhancer action, and suggest that RME is a consequence of general properties of gene regulation by enhancers rather than an RME-specific epigenetic program. Therefore, many and perhaps all genes may be subject to some degree of RME. Surprisingly, this was borne out by analysis of several genes that define different major hematopoietic lineages, that were previously thought to be universally expressed within those lineages: the genes encoding NKG2D, CD45, CD8α, and Thy-1. We propose that intrinsically probabilistic gene allele regulation is a general property of enhancer-controlled gene expression, with previously documented RME representing an extreme on a broad continuum.

MUTYH encodes a glycosylase involved in the base excision repair of DNA. Biallelic pathogenic germline variants in MUTYH cause an autosomal recessive condition known as MUTYH‐associated adenomatous polyposis and consequently increase the risk of colorectal cancer. However, reports of increased cancer risk in individuals carrying only one defective MUTYH allele are controversial and based on studies involving few individuals. Here, we describe a comprehensive investigation of monoallelic pathogenic MUTYH germline variants in 10,389 cancer patients across 33 different tumour types and 117,000 healthy individuals. Our results indicate that monoallelic pathogenic MUTYH germline variants can lead to tumorigenesis through a mechanism of somatic loss of heterozygosity of the functional MUTYH allele in the tumour. We confirmed that the frequency of monoallelic pathogenic MUTYH germline variants is higher in individuals with cancer than in the general population, although this frequency is not homogeneous among tumour types. We also demonstrated that the MUTYH mutational signature is present only in tumours with loss of the functional allele and found that the characteristic MUTYH base substitution (C>A) increases stop‐codon generation. We identified key genes that are affected during tumorigenesis. In conclusion, we propose that carriers of the monoallelic pathogenic MUTYH germline variant are at a higher risk of developing tumours, especially those with frequent loss of heterozygosity events, such as adrenal adenocarcinoma, although the overall risk is still low. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

How does the information in the genome program the functions of the wide variety of cells in the body? While the development of biological organisms appears to follow an explicit set of genomic instructions to generate the same outcome each time, many biological mechanisms harness molecular noise to produce variable outcomes. Non-deterministic variation is frequently observed in the diversification of cell surface molecules that give cells their functional properties, and is observed across eukaryotic clades, from single-celled protozoans to mammals. This is particularly evident in immune systems, where random recombination produces millions of antibodies from only a few genes; in nervous systems, where stochastic mechanisms vary the sensory receptors and synaptic matching molecules produced by different neurons; and in microbial antigenic variation. These systems employ overlapping molecular strategies including allelic exclusion, gene silencing by constitutive heterochromatin, targeted double-strand breaks, and competition for limiting enhancers. Here, we describe and compare five stochastic molecular mechanisms that produce variety in pathogen coat proteins and in the cell surface receptors of animal immune and neuronal cells, with an emphasis on the utility of non-deterministic variation.

Backgrounds: MUTYH‐associated polyposis (MAP) is an autosomal recessive disease caused by biallelic pathogenic variants (PV) of the MUTYH gene. The aim of this study was to investigate the genetic causes of unexplained polyposis patients with monoallelic MUTYH PV. The analysis focused on 26 patients with suspected MAP, belonging to 23 families. Ten probands carried also one or more additional MUTYH variants of unknown significance. Methods: Based on variant type and on the collected clinical and molecular data, these variants were reinterpreted by applying the ACMG/AMP rules. Moreover, supplementary analyses were carried out to investigate the presence of other variants and copy number variations in the coding and promoter regions of MUTYH, as well as other polyposis genes (APC, NTHL1, POLE, POLD1, MSH3, RNF43, and MCM9). Results: We reclassified 4 out of 10 MUTYH variants as pathogenic or likely pathogenic, thus supporting the diagnosis of MAP in only four cases. Two other patients belonging to the same family showed a previously undetected deletion of the APC gene promoter. No PVs were found in the other investigated genes. However, 6 out of the 18 remaining families are still interesting MAP candidates, due to the co‐presence of a class 3 MUTYH variant that could be reinterpreted in the next future. Conclusion: Several efforts are necessary to fully elucidate the genetic etiology of suspected MAP patients, especially those with the most severe polyposis/tumor phenotype. Clinical data, tumor molecular profile, family history, and polyposis inheritance mode may guide variant interpretation and address supplementary studies. [ABSTRACT FROM AUTHOR]

PurposeTo investigate the effect of ABCA4 mutation status on lipofuscin-related quantitative autofluorescence (qAF) in humans and on bisretinoid accumulation in mice.MethodsGenotyped parents (n = 26; age 37-64 years) of patients with biallelic ABCA4-related retinopathy underwent in-depth retinal phenotyping including qAF imaging as a surrogate measure for RPE lipofuscin accumulation. In addition, bisretinoids as the main components of autofluorescent lipofuscin at the ocular fundus were quantified in Abca4-/-, Abca4+/-, and wild-type mice.ResultsIndex patients showed a retinal phenotype characteristic for ABCA4-related retinopathy, including increased qAF levels. In contrast, qAF measures in carriers of only one ABCA4 mutation were not different from age-matched controls in this sample, and there was no difference between truncating and missense mutations. Also, none of these carriers presented an abnormal phenotype on conventional imaging. One parent with ABCA4-related retinopathy and increased qAF carried an additional ABCA4 mutation, explaining the phenotype under a recessive disease model (pseudodominance). Biochemical analysis in the mouse model revealed direct downstream products (A2PE-H2, at-RALdimer-PE) of the ABCA4 substrate N-Ret-PE to be similar in wild-type and Abca4+/- mice. Both bisretinoids were 12- to 18-fold increased in Abca4-/- mice. Levels of A2E and A2PE in Abca4+/- mice were in between those measured in wild-type and Abca4-/- mice.ConclusionsThis study indicates that carriers of monoallelic ABCA4 mutations are phenotypically normal. However, biochemical analysis in the Abca4-deficient mouse model suggests detectable effects of one mutation in ABCA4 on the molecular level. The findings may have implications for therapeutic approaches such as gene replacement therapy.

In diploid eukaryotic organisms, both alleles of each autosomal gene are usually assumed to be simultaneously expressed at similar levels. However, some genes can be expressed preferentially or strictly from a single allele, a process known as monoallelic expression. Classic monoallelic expression of X-chromosome-linked genes, olfactory receptor genes and developmentally imprinted genes is the result of epigenetic modifications. Genetic-origin-dependent monoallelic expression, however, is caused by cis-regulatory differences between the alleles. There is a paucity of systematic study to investigate these phenomena across multiple tissues, and the mechanisms underlying such monoallelic expression are not yet fully understood. Here we provide a detailed portrait of monoallelic gene expression across multiple tissues/cell lines in a hybrid mouse cross between the Mus musculus strain C57BL/6J and the Mus spretus strain SPRET/EiJ. We observed pervasive tissue-dependent allele-specific gene expression: in total, 1,839 genes exhibited monoallelic expression in at least one tissue, and 410 genes in at least two tissues. Among these 88 are monoallelic genes with different active alleles between tissues, probably representing genetic-origin-dependent monoallelic expression. We also identified six autosomal monoallelic genes with the active allele being identical in all eight tissues, which are likely novel candidates of imprinted genes. To depict the underlying regulatory mechanisms at the chromatin layer, we performed ATAC-seq in two different cell lines derived from the F1 mouse. Consistent with the global expression pattern, cell-type dependent monoallelic peaks were found, and a higher proportion of C57BL/6J-active peaks were observed in both cell types, implying possible species-specific regulation. Finally, only a small part of monoallelic gene expression could be explained by allelic differences in chromatin organization in promoter regions, suggesting that other distal elements may play important roles in shaping the patterns of allelic gene expression across tissues.

A mutation in the promoter of the Telomerase Reverse Transcriptase (TERT) gene is the most frequent noncoding mutation in cancer. The mutation drives unusual monoallelic expression of TERT, allowing immortalization. Here, we find that DNA methylation of the TERT CpG island (CGI) is also allele-specific in multiple cancers. The expressed allele is hypomethylated, which is opposite to cancers without TERT promoter mutations. The continued presence of Polycomb repressive complex 2 (PRC2) on the inactive allele suggests that histone marks of repressed chromatin may be causally linked to high DNA methylation. Consistent with this hypothesis, TERT promoter DNA containing 5-methyl-CpG has much increased affinity for PRC2 in vitro. Thus, CpG methylation and histone marks appear to collaborate to maintain the two TERT alleles in different epigenetic states in TERT promoter mutant cancers. Finally, in several cancers, DNA methylation levels at the TERT CGI correlate with altered patient survival.

Abstract Background Breast cancer (BC) is the most common type of cancer in women. Among many risk factors of BC, mutations in BRCA2 gene were found to be the primary cause in 5–10% of cases. The majority of deleterious mutations are frameshift or nonsense mutations. Most of the reported BRCA2 mutations are protein truncating mutations. Methods The study aimed to describe the pattern of mutations including single nucleotide polymorphisms (SNPs) and variants of the BRCA2 (exon11) gene among Sudanese women patients diagnosed with BC. In this study a specific region of BRCA2 exon 11 was targeted using PCR and DNA sequencing. Results Early onset cases 25/45 (55.6%) were premenopausal women with a mean age of 36.6 years. Multiparity was more frequent within the study amounting to 30 cases (66.6%), with a mean parity of 4.1. Ductal type tumor was the predominant type detected in 22 cases (48.8%) among the reported histotypes. A heterozygous monoallelic nonsense mutation at nucleotide 3385 was found in four patients out of 9, where TTA codon was converted into the stop codon TGA. Conclusion This study detected a monoallelic nonsense mutation in four Sudanese female patients diagnosed with early onset BC from different families. Further work is needed to demonstrate its usefulness in screening of BC.

The CRISPR technology continues to diversify with a broadening array of applications that touch all kingdoms of life. The simplicity, versatility and species-independent nature of the CRISPR system offers researchers a previously unattainable level of precision and control over genomic modifications. Successful applications in forest, fruit and nut trees have demonstrated the efficacy of CRISPR technology at generating null mutations in the first generation. This eliminates the lengthy process of multigenerational crosses to obtain homozygous knockouts (KO). The high degree of genome heterozygosity in outcrossing trees is both a challenge and an opportunity for genome editing: a challenge because sequence polymorphisms at the target site can render CRISPR editing ineffective; yet an opportunity because the power and specificity of CRISPR can be harnessed for allele-specific editing. Examination of CRISPR/Cas9-induced mutational profiles from published tree studies reveals the potential involvement of multiple DNA repair pathways, suggesting that the influence of sequence context at or near the target sites can define mutagenesis outcomes. For commercial production of elite trees that rely on vegetative propagation, available data suggest an excellent outlook for stable CRISPR-induced mutations and associated phenotypes over multiple clonal generations.

The CRISPR technology continues to diversify with a broadening array of applications that touch all kingdoms of life. The simplicity, versatility and species-independent nature of the CRISPR system offers researchers a previously unattainable level of precision and control over genomic modifications. Successful applications in forest, fruit and nut trees have demonstrated the efficacy of CRISPR technology at generating null mutations in the first generation. This eliminates the lengthy process of multigenerational crosses to obtain homozygous knockouts (KO). The high degree of genome heterozygosity in outcrossing trees is both a challenge and an opportunity for genome editing: a challenge because sequence polymorphisms at the target site can render CRISPR editing ineffective; yet an opportunity because the power and specificity of CRISPR can be harnessed for allele-specific editing. Examination of CRISPR/Cas9-induced mutational profiles from published tree studies reveals the potential involvement of multiple DNA repair pathways, suggesting that the influence of sequence context at or near the target sites can define mutagenesis outcomes. For commercial production of elite trees that rely on vegetative propagation, available data suggest an excellent outlook for stable CRISPR-induced mutations and associated phenotypes over multiple clonal generations. [ABSTRACT FROM AUTHOR]

Background: Breast cancer (BC) is the most common type of cancer in women. Among many risk factors of BC, mutations in BRCA2 gene were found to be the primary cause in 5-10% of cases. The majority of deleterious mutations are frameshift or nonsense mutations. Most of the reported BRCA2 mutations are protein truncating mutations. Methods: The study aimed to describe the pattern of mutations including single nucleotide polymorphisms (SNPs) and variants of the BRCA2 (exon11) gene among Sudanese women patients diagnosed with BC. In this study a specific region of BRCA2 exon 11 was targeted using PCR and DNA sequencing. Results: Early onset cases 25/45 (55.6%) were premenopausal women with a mean age of 36.6 years. Multiparity was more frequent within the study amounting to 30 cases (66.6%), with a mean parity of 4.1. Ductal type tumor was the predominant type detected in 22 cases (48.8%) among the reported histotypes. A heterozygous monoallelic nonsense mutation at nucleotide 3385 was found in four patients out of 9, where TTA codon was converted into the stop codon TGA. Conclusion: This study detected a monoallelic nonsense mutation in four Sudanese female patients diagnosed with early onset BC from different families. Further work is needed to demonstrate its usefulness in screening of BC. [ABSTRACT FROM AUTHOR]

This variant was previously identified as one allele in a French NBIA patient with another missense variant c.416 A>G;p.(Tyr139Cys) in compound heterozygous form, and in homozygous form in two affected MPAN patients [[5]], but no expression analyses were performed. Follow up of patients with monoallelic C19orf12 variants may reveal a second pathogenic variant in the same or a different gene. 1D: Partial sequence chromatograms indicating the wild type allele in a normal control, heterozygous family member and homozygous deletion mutation c.199delG;p.(Ala67LeufsTer5) in an affected member with C19orf12 variant. [Extracted from the article]

Allelic exclusion underpins antigenic variation and immune evasion in African trypanosomes. These bloodstream parasites use RNA polymerase-I (pol-I) to transcribe just one telomeric variant surface glycoprotein (VSG) gene at a time, producing superabundant and switchable VSG coats. We identified trypanosome VSG exclusion-1 (VEX1) using a genetic screen for defects in telomere-exclusive expression. VEX1 was sequestered by the active VSG and silencing of other VSGs failed when VEX1 was either ectopically expressed or depleted, indicating positive and negative regulation, respectively. Positive regulation affected VSGs and nontelomeric pol-I–transcribed genes, whereas negative regulation primarily affected VSGs. Negative regulation by VEX1 also affected telomeric pol-I–transcribed reporter constructs, but only when they contained blocks of sequence sharing homology with a pol-I–transcribed locus. We conclude that restricted positive regulation due to VEX1 sequestration, combined with VEX1-dependent, possibly homology-dependent silencing, drives a "winner-takes-all" mechanism of allelic exclusion. [ABSTRACT FROM AUTHOR]

Postnatal spermatogonial stem cells (SSCs) progress through proliferative and developmental stages to populate the testicular niche prior to productive spermatogenesis. To better understand, we conducted extensive genomic profiling at multiple postnatal stages on subpopulations enriched for particular markers (THY1, KIT, OCT4, ID4, or GFRa1). Overall, our profiles suggest three broad populations of spermatogonia in juveniles: (1) epithelial-like spermatogonia (THY1+; high OCT4, ID4, and GFRa1), (2) more abundant mesenchymal-like spermatogonia (THY1+; moderate OCT4 and ID4; high mesenchymal markers), and (3) (in older juveniles) abundant spermatogonia committing to gametogenesis (high KIT+). Epithelial-like spermatogonia displayed the expected imprinting patterns, but, surprisingly, mesenchymal-like spermatogonia lacked imprinting specifically at paternally imprinted loci but fully restored imprinting prior to puberty. Furthermore, mesenchymal-like spermatogonia also displayed developmentally linked DNA demethylation at meiotic genes and also at certain monoallelic neural genes (e.g., protocadherins and olfactory receptors). We also reveal novel candidate receptor-ligand networks involving SSCs and the developing niche. Taken together, neonates/juveniles contain heterogeneous epithelial-like or mesenchymal-like spermatogonial populations, with the latter displaying extensive DNA methylation/chromatin dynamics. We speculate that this plasticity helps SSCs proliferate and migrate within the developing seminiferous tubule, with proper niche interaction and membrane attachment reverting mesenchymal-like spermatogonial subtype cells back to an epithelial-like state with normal imprinting profiles. [ABSTRACT FROM AUTHOR]

Somatic mutations in the promoter of the gene for telomerase reverse transcriptase (TERT) are the most common noncoding mutations in cancer. They are thought to activate telomerase, contributing to proliferative immortality, but the molecular events driving TERT activation are largely unknown. We observed in multiple cancer cell lines that mutant TERT promoters exhibit the H3K4me2/3 mark of active chromatin and recruit the GABPA/B1 transcription factor, while the wild-type allele retains the H3K27me3 mark of epigenetic silencing; only the mutant promoters are transcriptionally active. These results suggest how a single-base-pair mutation can cause a dramatic epigenetic switch and monoallelic expression. [ABSTRACT FROM AUTHOR]

Studies on Variant Surface Glycoproteins (VSGs) and antigenic variation in the African trypanosome, Trypanosoma brucei, have yielded a remarkable range of novel and important insights. The features first identified in T. brucei extend from unique to conserved-among-trypanosomatids to conserved-among-eukaryotes. Consequently, much of what we now know about trypanosomatid biology and much of the technology available has its origin in studies related to VSGs. T. brucei is now probably the most advanced early branched eukaryote in terms of experimental tractability and can be approached as a pathogen, as a model for studies on fundamental processes, as a model for studies on eukaryotic evolution or often all of the above. In terms of antigenic variation itself, substantial progress has been made in understanding the expression and switching of the VSG coat, while outstanding questions continue to stimulate innovative new approaches. There are large numbers of VSG genes in the genome but only one is expressed at a time, always immediately adjacent to a telomere. DNA repair processes allow a new VSG to be copied into the single transcribed locus. A coordinated transcriptional switch can also allow a new VSG gene to be activated without any detectable change in the DNA sequence, thereby maintaining singular expression, also known as allelic exclusion. I review the story behind VSGs; the genes, their expression and switching, their central role in T. brucei virulence, the discoveries that emerged along the way and the persistent questions relating to allelic exclusion in particular. [ABSTRACT FROM AUTHOR]

When pluripotent cells are exposed to a uniform culture environment they routinely display heterogeneous gene expression. Aspects of this heterogeneity, such as Nanog expression, are linked to differences in the propensity of individual cells to either self-renew or commit towards differentiation. Recent findings have provided new insight into the underlying causes of this heterogeneity, which we summarise here using Nanog, a key regulator of pluripotency, as a model gene. We discuss the role of transcription factor heterogeneity in facilitating the intrinsically dynamic and stochastic nature of the pluripotency network, which in turn provides a potential benefit to a population of cells that needs to balance cell fate decisions. [ABSTRACT FROM AUTHOR]

The generation of complex neural circuits depends on the correct wiring of neurons with diverse individual characteristics. To understand the complexity of the nervous system, the molecular mechanisms for specifying the identity and diversity of individual neurons must be elucidated. The clustered protocadherins (Pcdh) in mammals consist of approximately 50 Pcdh genes (Pcdh-α, Pcdh-β, and Pcdh-γ) that encode cadherin-family cell surface adhesion proteins. Individual neurons express a random combination of Pcdh-α and Pcdh-γ, whereas the expression patterns for the Pcdh-β genes, 22 one-exon genes in mouse, are not fully understood. Here we show that the Pcdh-β genes are expressed in a 3’-polyadenylated form in mouse brain. In situ hybridization using a pan-Pcdh-β probe against a conserved Pcdh-β sequence showed widespread labeling in the brain, with prominent signals in the olfactory bulb, hippocampus, and cerebellum. In situ hybridization with specific probes for individual Pcdh-β genes showed their expression to be scattered in Purkinje cells from P10 to P150. The scattered expression patterns were confirmed by performing a newly developed single-cell 3’-RACE analysis of Purkinje cells, which clearly demonstrated that the Pcdh-β genes are expressed monoallelically and combinatorially in individual Purkinje cells. Scattered expression patterns of individual Pcdh-β genes were also observed in pyramidal neurons in the hippocampus and cerebral cortex, neurons in the trigeminal and dorsal root ganglion, GABAergic interneurons, and cholinergic neurons. Our results extend previous observations of diversity at the single-neuron level generated by Pcdh expression and suggest that the Pcdh-β cluster genes contribute to specifying the identity and diversity of individual neurons.

Losses in 13q as a sole abnormality confer a good prognosis in chronic lymphocytic leukaemia ( CLL). Nevertheless, its heterogeneity has been demonstrated and the clinical significance of biallelic 13q deletions remains controversial. We compared the clinico-biological characteristics of a series of 627 patients harbouring isolated 13q deletions by fluorescence in situ hybridization ( FISH), either monoallelic (13q × 1), biallelic (13q × 2), or the coexistence of both clones (13q M). The most frequent 13q deletion was 13q × 1 (82·1%), while 13q × 2 and 13q M represented 8·6% and 9·3% of patients respectively. The median percentage of altered nuclei significantly differed across groups: 55%, 72·5% and 80% in 13q × 1, 13q × 2 and 13qM ( P < 0·001). However, no significant differences in the clinical outcome among 13q groups were found. From 84 patients with sequential FISH studies, eight patients lost the remaining allele of 13q whereas none of them changed from 13q × 2 to the 13q × 1 group. The percentage of abnormal cells detected by FISH had a significant impact on the five-year cumulative incidence of treatment and the overall survival, 90% being the highest predictive power cut-off. In conclusion, loss of the remaining 13q allele is not enough to entail a worse prognosis in CLL. The presence of isolated 13q deletion can be risk-stratified according to the percentage of altered cells. [ABSTRACT FROM AUTHOR]

BACKGROUND: Fluorescence in situ hybridization can detect genomic abnormalities in up to 80% of cases and provides prognostic information on patients with chronic lymphocytic leukemia (CLL). Although 13q deletion as the sole abnormality has been found to confer a favorable prognosis, there are little data as to whether there is a difference in prognostic value between monoallelic versus biallelic deletion of 13q. METHODS: The authors reviewed the electronic database for patients with CLL who carried the 13q deletion as the sole abnormality and presented to The University of Texas MD Anderson Cancer Center (MDACC). Untreated patients were separated into 2 groups: those having monoallelic versus those with biallelic deletion of 13q. Using Mann-Whitney, chi-square, and Kaplan-Meier analysis, the baseline quantitative and qualitative variables for each group, along with the time from presentation to MDACC to treatment, were compared. RESULTS: A total of 176 patients were identified; 143 patients had a monoallelic deletion of 13q, whereas 33 patients had a biallelic deletion. The only significantly different values between the groups were albumin (4.5 g/dL vs 4.4 g/dL; P = .01) and zeta-chain-associated protein kinase 70 (ZAP70) expression (1.7% vs 4.8%; P = .010). The median time from fluorescence in situ hybridization analysis to treatment in both the monoallelic and biallelic groups had not been reached ( P = not significant). CONCLUSIONS: Except for inconsequential differences in albumin and ZAP70 expression, there was no difference in the baseline characteristics between patients with CLL who had monoallelic or biallelic deletion of 13q. In addition, there was no significant difference in endpoints, including time to treatment. Cancer 2012;3531-3537. © 2011 American Cancer Society. [ABSTRACT FROM AUTHOR]

Loss of a critical region in 13q14.3 [del(13q)] is the most common genomic aberration in chronic lymphocytic leukemia (CLL), occurring in more than 50% of patients (Stilgenbauer et al., Oncogene 1998;16:1891 - 1897, Dohner et al., N Engl J Med 2000;343:1910 - 1916). Despite extensive investigations, no point mutations have been found in the remaining allele that would inactivate one of the candidate tumor suppressor genes and explain the pathomechanism postulated for this region. However, the genes in the region are significantly down-regulated in CLL cells, more than would be expected by gene dosage, and recently a complex epigenetic regulatory mechanism was identified for 13q14.3 in non-malignant cells that involves asynchronous replication timing and monoallelic expression of candidate tumor suppressor genes. Here, we propose a model of a multigenic pathomechanism in 13q14.3, where several tumor suppressor genes, including the miRNA genes miR-16-1 and miR-15a, are co-regulated by the two long non-coding RNA genes DLEU1 and DLEU2 that span the critical region. Furthermore, we propose these co-regulated genes to be involved in the same molecular pathways, thereby also forming a functional gene cluster. Elucidating the molecular and cellular function of the 13q14.3 candidate genes will shed light on the underlying pathomechanism of CLL. [ABSTRACT FROM AUTHOR]

Abstract: There are two functional insulin genes in the mouse genome. The Ins2 gene is imprinted and expressed monoallelically from the paternal allele in the yolk sac. In the present study we have re-examined the imprinting status of Ins1. We found that Ins1 is not expressed in the yolk sac of several laboratory mouse strains. The asynchrony of replication at the wild type locus was significantly lower than at imprinted loci and was more similar to non-imprinted loci. Finally, we have taken the advantage of the Ins1neo allele created by homologous recombination to examine the allelic usage at this locus. We observed that the neo gene inserted at the Ins1 locus was expressed from both the paternally and the maternally transmitted allele. Therefore, the Ins1 gene does not share any of the basic properties of imprinted genes. On the basis of these data, we concluded that Ins1 locus is unlikely to be imprinted in common laboratory mice. [Copyright &y& Elsevier]

Bernard-Soulier syndrome (BSS) is a rare inherited disorder characterized by unusually large platelets, low platelet count, and prolonged bleeding time. BSS is usually inherited in an autosomal recessive (AR) mode of inheritance due to a deficiency of the GPIb-IX-V complex also known as the von Willebrand factor (VWF) receptor. We investigated a family with macrothrombocytopenia, a mild bleeding tendency, slightly lowered platelet aggregation tests, and suspected autosomal dominant (AD) inheritance. We have detected a heterozygous GP1BA likely pathogenic variant, causing monoallelic BSS. A germline GP1BA gene variant (NM_000173:c.98G > A:p.C33Y), segregating with the macrothrombocytopenia, was detected by whole-exome sequencing. In silico analysis of the protein structure of the novel GPIbα variant revealed a potential structural defect, which could impact proper protein folding and subsequent binding to VWF. Flow cytometry, immunoblot, and electron microscopy demonstrated further differences between p.C33Y GP1BA carriers and healthy controls. Here, we provide a detailed insight into its clinical presentation and phenotype. Moreover, the here described case first presents an mBSS patient with two previous ischemic strokes. [ABSTRACT FROM AUTHOR]

Simple Summary: The importance of gene variants in the prognosis of myelodysplastic syndromes (MDSs) has been repeatedly reported in recent years. Especially, TP53 mutations are independently associated with a higher risk category, resistance to conventional therapies, rapid transformation to leukemia, and a poor outcome. In the review, we discuss the features of monoallelic and biallelic TP53 mutations within MDS, the carcinogenic mechanisms, and the predictive value of TP53 variants in current standard treatments including hypomethylating agents, allogeneic hematopoietic stem cell transplantation, and lenalidomide, as well as the latest progress in TP53-targeted therapy strategies in MDS. Myelodysplastic syndromes (MDSs) are heterogeneous for their morphology, clinical characteristics, survival of patients, and evolution to acute myeloid leukemia. Different prognostic scoring systems including the International Prognostic Scoring System (IPSS), the Revised IPSS, the WHO Typed Prognostic Scoring System, and the Lower-Risk Prognostic Scoring System have been introduced for categorizing the highly variable clinical outcomes. However, not considered by current MDS prognosis classification systems, gene variants have been identified for their contribution to the clinical heterogeneity of the disease and their impact on the prognosis. Notably, TP53 mutation is independently associated with a higher risk category, resistance to conventional therapies, rapid transformation to leukemia, and a poor outcome. Herein, we discuss the features of monoallelic and biallelic TP53 mutations within MDS, their corresponding carcinogenic mechanisms, their predictive value in current standard treatments including hypomethylating agents, allogeneic hematopoietic stem cell transplantation, and lenalidomide, together with the latest progress in TP53-targeted therapy strategies, especially MDS clinical trial data. [ABSTRACT FROM AUTHOR]

We developed an in vitro system to differentiate embryonic stem cells (ESCs) derived from reciprocally crossed F1 hybrid mice into neurons, and used it to investigate poly(A)+ and total RNA transcription at different stages of cell differentiation. By comparing expression profiles of transcripts assembled from 20 RNA sequencing datasets [2 alleles×(2 cell lines×4 time-points+2 mouse brains)], the relative influence of strain, cell and parent specificities to overall expression could be assessed. Divergent expression profiles of ESCs converged tightly at neural progenitor stage. Patterns of temporal variation of monoallelically expressed transcripts and antisense transcripts were quantified. Comparison of sense and antisense transcript pairs within the poly(A)+ sample, within the total RNA sample, and across poly(A)+ and total RNA samples revealed distinct rates of pairs showing anti-correlated expression variation. Unique patterns of sharing of poly(A)+ and poly(A)- transcription were identified in distinct RNA species. Regulation and functionality of monoallelic expression, antisense transcripts and poly(A)- transcription remain elusive. We demonstrated the effectiveness of our approach to capture these transcriptional activities, and provided new resources to elucidate the mammalian developmental transcriptome., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Summary Interactions between genetic and epigenetic effects shape brain function, behavior, and the risk for mental illness. Random X inactivation and genomic imprinting are epigenetic allelic effects that are well known to influence genetic architecture and disease risk. Less is known about the nature, prevalence, and conservation of other potential epigenetic allelic effects in vivo in the mouse and primate brain. Here we devise genomics, in situ hybridization, and mouse genetics strategies to uncover diverse allelic effects in the brain that are not caused by imprinting or genetic variation. We found allelic effects that are developmental stage and cell type specific, that are prevalent in the neonatal brain, and that cause mosaics of monoallelic brain cells that differentially express wild-type and mutant alleles for heterozygous mutations. Finally, we show that diverse non-genetic allelic effects that impact mental illness risk genes exist in the macaque and human brain. Our findings have potential implications for mammalian brain genetics. Video Abstract [ABSTRACT FROM AUTHOR]

Common terms used in statistical genetics with multiple meanings are explained and the terminology used in subsequent chapters is defined. Statistical human genetics has existed as a discipline for over a century, and during that time the meanings of many of the terms used have evolved, largely driven by molecular discoveries, to the point that molecular geneticists, statistical geneticists, and statisticians often have difficulty understanding each other. It is therefore imperative, now that so much of molecular genetics is becoming an in silico and statistical science, that we have a well-defined, common terminology.